US2012122700A1PendingUtilityA1

Materials and methods for determining subtelomere dna sequence

25
Individually held — no corporate assignee on recordPriority: Nov 11, 2010Filed: Nov 14, 2011Published: May 17, 2012
Est. expiryNov 11, 2030(~4.3 yrs left)· nominal 20-yr term from priority
Inventors:Peter G. MedveczkyMaria M. MedveczkyJesse H. Arbuckle
C12Q 1/705
25
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Claims

Abstract

The subject invention pertains to methods for rapid and accurate determination of subtelomere DNA sequences. Also provided are kits for determination of subtelomere sequences and uses of chromosomal terminal sequences for studying pathogenesis and treatment of diseases.

Claims

exact text as granted — not AI-modified
1 . A method for determining a subtelomere DNA sequence of a host, comprising:
 a) providing a population of host cells that carry HHV-6 genomic DNA in host chromosomal DNA;   b) subjecting the host chromosomal DNA to inverse polymerase chain reaction (IPCR), thereby generating HHV-6-subtelomere DNA; and   c) determining the HHV-6-subtelomere DNA sequence.   
     
     
         2 . The method according to  claim 1 , comprising, after step a), the step of: extracting chromosomal DNA from host cells. 
     
     
         3 . The method according to  claim 1 , comprising determining the host chromosome that carries HHV-6-subtelomere DNA using fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), fluorescent labeling of HHV-6 DNA, Southern blot, or any combination thereof. 
     
     
         4 . The method according to  claim 3 , wherein the host chromosome that carries HHV-6-subtelomere DNA is determined by fluorescence in situ hybridization (FISH) using HHV-6-specific DNA probes. 
     
     
         5 . The method according to  claim 3 , wherein the host chromosome that carries HHV-6-subtelomere DNA is determined by Southern Blot using HHV-6-specific and/or chromosome-specific probes. 
     
     
         6 . The method according to  claim 1 , wherein step b) comprises:
 digesting the host chromosomal DNA using restriction endonuclease molecules, thereby generating host chromosomal DNA fragments comprising the HHV-6-subtelomere DNA; and   circularizing the fragments comprising the HHV-6-subtelomere DNA via intramolecular ligation.   
     
     
         7 . The method according to  claim 6 , wherein step b) further comprises: diluting host chromosomal DNA fragments after restriction digestion. 
     
     
         8 . The method according to  claim 6 , wherein the restriction endonuclease molecule is selected from the group consisting of MboI, Mme I, HaeIII, FspBI, Csp6I, NciI, NsiI, PovII, Esp3I, PstII, ClaI, BssHII, TaqI, RsaI, Sau3AI, HindIII, EcoRI, EcoRII, BamHI, NotI, SamI, AluI, KpnI, PstI, ScaI, and XbaI. 
     
     
         9 . The method according to  claim 8 , wherein the restriction endonuclease molecule is MboI. 
     
     
         10 . The method according to  claim 1 , wherein the host cells carry HHV-6A DNA, HHV-6B DNA, or both. 
     
     
         11 . The method according to  claim 10 , wherein the host cells carry HHV-6A strain U1102 or HHV-6B Z29 strain. 
     
     
         12 . The method according to  claim 12 , wherein the HHV-6-subtelomere DNA is generated using an IPCR primer that is complementary to at least 15 contiguous nucleotides of HHV-6 right end direct repeat (DR R ) DNA of any of U1-U100. 
     
     
         13 . The method according to  claim 1 , wherein the IPCR primer is complementary to at least 15 contiguous nucleotides of any of HHV-6 U53, HHV-6-U54 and HHV-6-U94 DR R  DNA. 
     
     
         14 . The method according to  claim 12 , wherein the IPCR primer comprises SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         15 . The method according to  claim 1 , wherein the sequence of the HHV-6-subtelomere DNA is determined using dideoxy sequencing reactions (Sanger sequencing), sequencing by synthesis using reversibly terminated labeled nucleotides, primer walking, shotgun sequencing, gel electrophoresis sequencing, multi-color fluorescence-based DNA techniques, sequencing by hybridization, DNA microarray, pyrosequencing, 454 sequencing, polony sequencing, SOLiD sequencing, or any combination thereof. 
     
     
         16 . The method according to  claim 1 , wherein the chromosome of interest is of mammalian origin. 
     
     
         17 . The method according to  claim 1 , wherein the host cells are isolated from an HHV-6-infected subject, and wherein the sequence of the HHV-6-subtelomere DNA indicates the host chromosomal telomeric site into which HHV-6 DNA is inserted. 
     
     
         18 . A kit for determining the subtelomere DNA sequence of a chromosome of interest, comprising: an IPCR primer that is, or is complementary to, at least 15 contiguous nucleotides of one strand HHV-6 right end direct repeat (DR R ) sequence of any of U1-U100. 
     
     
         19 . The kit according to  claim 18 , wherein the IPCR primer comprises SEQ ID NO:1 (IPCR-1) or SEQ ID NO:2 (IPCR-2). 
     
     
         20 . The kit according to  claim 18 , further comprising one or more of materials selected from the group consisting of primer for host chromosomal-specific PCR, host chromosomal-specific hybridization probe, hybridization probe for identifying HHV-6 DNA, hybridization probe for identifying host telomere DNA, hybridization probe for identifying host subtelomere DNA, restriction enzyme molecules, DNA ligase, and Taq DNA polymerase. 
     
     
         21 . The kit according to  claim 18 , comprising oligonucleotide molecules comprising any of SEQ ID NO:3-SEQ ID NO:40.

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